The activity-regulated cytoskeletal-associated protein (Arc, also known as Arg3.1) is an immediate early gene product induced by activity/experience and required multiple modes of synaptic plasticity. Both long-term potentiation (LTP) and long-term depression (LTD) are impaired upon Arc deletion, as well as the ability to form long-term spatial, taste and fear memories. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors (AMPARs) in dendritic spines, a process associated with LTD. This role for Arc in AMPAR endocytosis was supported by a report that Arc binds directly to two elements of the endocytic machinery, dynamin and endophilin, and by our finding that Arc stimulates dynamin self-assembly and GTPase activity. Solution of the crystal structure of a C-terminal segment of Arc revealed a striking similarity to the capsid domain of HIV Gag. Moreover, Arc assembles into viral capsid-like structures that enclose Arc mRNA, are released into the extracellular space, and are internalized by neighboring cells. Thus, Arc is unique in promoting plasma membrane budding both into and out of the cell. The goal of this project is to define the mechanism and regulation of Arc-mediated membrane vesiculation. Fluorescence fluctuation spectroscopy (FFS) will be utilized to monitor membrane budding giant unilamellar vesicles (GUVs). In Aim 1 we will use spectral phasor analysis to determine how lipid composition and fluidity influence the budding process, and Number and Brightness (N&B) analysis to obtain a quantitative picture of Arc self-assembly on the GUV surface. In Aim 2 we will determine how post-translational modifications of Arc, recently identified in our laboratories, control the binding of Arc to lipids and mRNA and Arc-mediated membrane vesiculation. Changes in Arc expression have been linked to numerous cognitive disorders, including mental retardation, Alzheimer's Disease, and substance abuse. Therefore, elucidation of mechanisms that regulate Arc-mediated intercellular communication has potential clinical significance.